Ever since Ronaghi invented pyrosequencing method, opening up the era of second generation sequencing, up to now the second generation sequencing has been experiencing a phase of rapid development. However, with the advances in high-throughput sequencing, the aspect of high-throughput and low-cost sample preparation is becoming a key factor of great concern. Sample treatment methods and automatic apparatuses based on various principles have been continually developed, which mainly include sample fragmentation, terminal treatment of nucleic acid molecules, and adaptor ligation, etc.
Sample fragmentation is mainly achieved by physical methods such as ultrasonic cleaving, or enzymatic methods such as non-specific endonuclease treatment. The physical method predominantly used involves a Covaris disruptor based on proprietary Adaptive Focused Acoustics (AFA) technology, whereby geometrically focused acoustic energy is utilized under isothermal condition. Acoustic energy having a wavelength of 1 mm is focused to a sample by a spherical solid-state ultrasonic transducer of >400 kHz. The method ensures the maintenance of the completeness of nucleic acid samples and achieves a high recovery rate. The Covaris disruptor includes the economical M series, the single-tube full-power S series and the higher throughput E and L series. The fragments obtained from the physical method exhibit a good fragment randomness. However, a number of Covaris disruptors are needed for the sake of throughput, and subsequent separate operations of terminal processing, adaptor ligation, PCR and various purifications are also required. One of the enzymatic methods involves NEB Next dsDNA Fragmentase available from NEB. The reagent first introduces random nick positions into double-stranded DNA, then recognizes the nick positions and cleaves the complementary DNA strands with another enzyme, thus achieving the aim of disrupting the DNA. Such a reagent can be used in genome DNA, whole genome amplification products, PCR products etc. and provides good randomness. Nevertheless, it will generate some artificial short fragment insertions and deletions, and also inevitably entails subsequent separate operations of terminal processing, adaptor ligation, PCR and corresponding purifications. Additionally, the transposase disruption kits available from Epicentra, as represented by Nextera kit, utilize transposase to achieve DNA fragmentation and adaptor addition at the same time, thus reducing the length of time for sample processing.
Considering the convenience of various operations, the transposase disruption method is undoubtedly much better than other methods in terms of throughput and convenience in operation. Nevertheless, such a disruption method has its own disadvantage as follows. The transposase relies on a specific 19 bp Me sequence to achieve transposition. Therefore, although the transposase can embed two completely different adaptor sequences and thus add the different adaptor sequences respectively to the 5′-end and the 3′-end of a target sequence, both adaptors need to comprise a specific Me sequence. One of the resulting influences is that the fragments generated from disruption will have, symmetrically, a Me sequence on both terminal ends. Also, there will be a gap of 9 nt base deletion between the sequence of interest or the sequence obtained from disruption and the Me sequence, due to the special effect of the transposase. Identical Me sequences franking the target sequence may affect some of the downstream applications. For example, with respect to the second generation sequencing technology based on ligation process, the Me sequences flanking the same strand are complementary sequences, which would easily result in annealing within the single-stranded molecule and is thus unfavorable for the binding of the anchoring primers.
Chinese patent application CN 102703426 A proposes a method to address this issue, in which the sequences obtained following disruption are subjected to digestion with a specific endonuclease to remove the 9 nt sequence and the Me sequence. This method only takes advantage of transposase disruption to randomly disrupt a nucleic acid sequence, but introduces the drawback that subsequently adaptors need to be added separately. Also, the method suffers from excessive steps and is not adapted to applications of higher throughput.